Cloning And Characterization Of OsMPK4, A Mitogen-activated Protein Kinase Gene, From Rice

Mitogen-activated protein kinase (MAPK) cascades play important roles in development, cell proliferation and hormone response, as well as in biotic and abiotic stress signaling pathoways in plants. In this study, we cloned and identified a novel rice MAPK gene, 0sMPK4 based on the conserved subdomains of MAPKs. The full-length cDNA of 0sMPK4 consists of 1483 bp in size, containing an open reading frame of 1131 bp, which predicts to encode a protein of 376 amino acids with calculated molecular weight of 42.8 kDa. The OsMPK4 gene is located on chromosome 10 of the rice genome and consists of 6 exons and 5 introns. The OsMPK4 protein contains all 11 conserved domains that are characteristics of MAPKs and phosphorylation site of TEY motif. Phylogenetic tress analysis revealed that OsMPK4 belongs to Group B of the plant MAPKs;however, OsMPK4 shows a relatively low level of identity to all other characterized rice MAPKs, indicating that OsMPK4 is a new MAPK gene in rice.To better understand the function of OsMPK4 in plant disease resistance, we cloned the coding region of OsMPK4 into plant binary vector pCHF3, under control of CaMV 35S promoter and transformed tobacco leaf discs by Agrobacterium-media.ted transformation. Twenty-one kanamycin-resistant transgneic tobacco lines were obtained and confirmed by PCR detetion. Preliminary results showed that the transgenic lines overexpressing OsMPK4 have some morphological abnormalities and reduced fertility. Studies on disease resistance and other phenotype of the transgenic tobacco lines overexpressing OsMPK4 are in progress.To characterize the biochemical function of OsMPK4, the OsMPK4 gene was cloned into the pRSET-A vector to create a recombinant plasmid pRSETA-OsMPK4. Recombinant OsMPK4 protein expressed in E. coli was purified, which will be used to study OsMPK4 biochemical activity.